Methods for preserving organs and tissues

ABSTRACT

The invention relates to a method for preserving an organ or tissue comprising contacting the organ or tissue with an effective amount of a kallikrein inhibitor and solutions useful for such a method. Also provided is a method for reducing reperfusion injury of an organ during surgery and/or following removal of the organ from a subject comprising placing the organ in an organ storage and preservative solution, wherein the solution comprises a kallikrein inhibitor.

RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/407,004, filed Aug. 28, 2002.

[0002] The entire teachings of the above application are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0003] Preservation of the viability of donor organs is an importantgoal for organ transplantation. Typically the organ to be transplantedmust be stored and shipped to the prospective recipient. The ability toprolong the cellular viability of the organ during storage andtransportation is very important to the success of the transplantoperation. Preservative solutions play an important role in thelongevity of the organ. Solutions for organ preservation include thosedescribed by Berdyaev et al., U.S. Pat. No. 5,432,053; Belzer et al.,U.S. Pat. Nos. 4,798,824, 4,879,283, and 4,873,230; Taylor, U.S. Pat.No. 5,405,742; Dohi et al., U.S. Pat. No. 5,565,317; Stern et al., U.S.Pat. Nos. 5,370,989 and 5,552,267, the contents of which areincorporated herein by reference in their entirety. However, a needexists for improved methods and solutions for organ preservation.

[0004] Proteases are involved in a broad range of biological pathways.In particular, serine proteases such as kallikrein, plasmin, elastase,urokinase plasminogen activator, thrombin, human lipoprotein-associatedcoagulation inhibitor, and coagulation factors such as factors VIIa,IXa, Xa, XIa, and XIIa have been implicated in pathways affecting bloodflow, e.g., general and focal ischemia, tumor invasion, fibrinolysis,perioperative blood loss, and inflammation. Inhibitors of specificserine proteases, therefore, have received attention as potential drugtargets for various ischemic maladies.

[0005] One such inhibitor, aprotinin (also called bovine pancreatictrypsin inhibitor or BPTI), obtained from bovine lung, has been approvedin the United States for prophylactic use in reducing perioperativeblood loss and the need for transfusion in patients undergoing CPB,e.g., in the course of a coronary artery bypass grafting procedure.Aprotinin is commercially available under the trade name TRASYLOL®(Bayer Corporation Pharmaceutical Division, West Haven, Conn.) and waspreviously approved for use to treat pancreatitis. The effectiveness ofaprotinin is associated with its relatively non-specific abilities toinhibit a variety of serine proteases, including plasma kallikrein, andplasmin. These proteases are important in a number of pathways of thecontact activation system (CAS).

[0006] CAS is initially activated when whole blood contacts the surfaceof foreign substrates (e.g., kaolin, glass, dextran sulfate, or damagedbone surfaces). Kallikrein, a serine protease, is a plasma enzyme thatinitiates the CAS cascade leading to activation of neutrophils, plasmin,coagulation, and various kinins. Kallikrein is secreted as a zymogen(pre-kallikrein) that circulates as an inactive molecule until activatedby a proteolytic event early in the contact activation cascade.

[0007] However, the use of specific kallikrein inhibitors for organpreservation has not been successfully demonstrated.

SUMMARY OF THE INVENTION

[0008] This invention is based on the discovery of peptides that inhibitserine proteases, such as, for example, kallikrein, which cansuccessfully be employed to preserve an organ pending transplant. Morespecifically, the invention provides methods of using kallikreininhibitors in a method for preserving an organ or tissue andcompositions for such use. The invention also relates to methods forreducing, inhibiting or preventing reperfusion injury or damage in anorgan or tissue that has been removed from its host and compositions forsuch use. Preferred kallikrein peptides include those described in U.S.Pat. Nos. 6,333,402 and 6,057,287 to Markland et al., the contents ofwhich are incorporated herein by reference in their entirety.

[0009] In a particularly preferred embodiment, the invention is directedto compositions comprising a polypeptide comprising the amino acidsequence: (SEQ ID NO:1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 PheXaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58,

[0010] wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are eachindividually an amino acid or absent; Xaa6, Xaa7, Xaa8, Xaa9, Xaa20,Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41, Xaa42, Xaa44, Xaa46,Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54 can be any aminoacid; Xaa10 is an amino acid selected from the group consisting of: Aspand Glu; Xaa11 is an amino acid selected from the group consisting of:Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr; Xaa13 is an amino acidselected from the group consisting of: Arg, His, Pro, Asn, Ser, Thr,Ala, Gly, Lys and Gln; Xaa15 is an amino acid selected from the groupconsisting of: Arg, Lys, Ala, Ser, Gly, Met, Asn and Gln; Xaa16 is anamino acid selected from the group consisting of: Ala, Gly, Ser, Asp andAsn; Xaa17 is an amino acid selected from the group consisting of: Ala,Asn, Ser, Ile, Gly, Val, Gln and Thr; Xaa18 is an amino acid selectedfrom the group consisting of: His, Leu, Gln and Ala; Xaa19 is an aminoacid selected from the group consisting of: Pro, Gln, Leu, Asn and Ile;Xaa21 is an amino acid selected from the group consisting of: Trp, Phe,Tyr, His and Ile; Xaa22 is an amino acid selected from the groupconsisting of: Tyr and Phe; Xaa23 is an amino acid selected from thegroup consisting of: Tyr and Phe; Xaa31 is an amino acid selected fromthe group consisting of: Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile andThr; Xaa32 is an amino acid selected from the group consisting of: Glu,Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly and Val; Xaa34 is an aminoacid selected from the group consisting of: Thr, Ile, Ser, Val, Ala,Asn, Gly and Leu; Xaa35 is an amino acid selected from the groupconsisting of: Tyr, Trp and Phe; Xaa39 is an amino acid selected fromthe group consisting of: Glu, Gly, Ala, Ser and Asp; Xaa40 is an aminoacid selected from the group consisting of: Gly and Ala; Xaa43 is anamino acid selected from the group consisting of: Asn and Gly; Xaa45 isan amino acid selected from the group consisting of: Phe and Tyr; andwherein said polypeptide inhibits kallikrein, and methods of using suchcompositions.

[0011] In a particular embodiment, specific amino acid positions can bethe following: Xaa6 can be Ala, Xaa7 can be Phe, Xaa8 can be Lys, Xaa9can be Ala, Xaa10 can be Asp, Xaa11 can be Asp, Xaa13 can be Pro, Xaa15can be Arg, Xaa16 can be Ala, Xaa17 can be Ala, Xaa18 can be His, Xaa19can be Pro, Xaa20 can be Arg, Xaa24 can be Asn, Xaa25 can be Ile, Xaa26can be Phe, Xaa27 can be Thr, Xaa28 can be Arg, Xaa29 can be Gln, Xaa31can be Glu, Xaa32 can be Glu, Xaa34 can be Ile, Xaa35 can be Tyr, Xaa39can be Glu, Xaa41 can be Asn, Xaa42 can be Arg, Xaa44 can be Arg, Xaa46can be Glu, Xaa47 can be Ser, Xaa48 can be Leu, Xaa49 can be Glu, and/orXaa50 can be Glu; any of these specific amino acids at these positionscan occur individually or in combination with one or more of the aminoacids at one or more position otherwise described.

[0012] In a particular embodiment, the present invention is directed toa composition comprising a polypeptide as described in SEQ ID NO:1, suchthat two or more of the following amino acid positions are defined asfollows: Xaa10 can be Asp; Xaa11 can be Asp; Xaa13 can be Pro; Xaa15 canbe Arg; Xaa16 can be Ala; Xaa17 can be Ala; Xaa18 can be His; Xaa19 canbe Pro; Xaa21 can be Trp; Xaa22 can be Phe; Xaa23 can be Phe; Xaa31 canbe Glu; Xaa32 can be Glu; Xaa34 can be Ile; Xaa35 can be Tyr; Xaa39 canbe Glu; Xaa40 can be Gly; Xaa43 can be Asn; and Xaa45 can be Phe, andmethods of using such compositions. In another embodiment, five or moreof the following of the following amino acid positions are defined asfollows: Xaa10 can be Asp; Xaa11 can be Asp; Xaa13 can be Pro; Xaa15 canbe Arg; Xaa16 can be Ala; Xaa17 can be Ala; Xaa18 can be His; Xaa19 canbe Pro; Xaa21 can be Trp; Xaa22 can be Phe; Xaa23 can be Phe; Xaa31 canbe Glu; Xaa32 can be Glu; Xaa34 can be Ile; Xaa35 can be Tyr; Xaa39 canbe Glu; Xaa40 can be Gly; Xaa43 can be Asn; and Xaa45 can be Phe. Inanother embodiment, 10 or more of the amino acids are defined asfollows: Xaa10 can be Asp; Xaa11 can be Asp; Xaa13 can be Pro; Xaa15 canbe Arg; Xaa16 can be Ala; Xaa17 can be Ala; Xaa18 can be His; Xaa19 canbe Pro; Xaa21 can be Trp; Xaa22 can be Phe; Xaa23 can be Phe; Xaa31 canbe Glu; Xaa32 can be Glu; Xaa34 can be Ile; Xaa35 can be Tyr; Xaa39 canbe Glu; Xaa40 can be Gly; Xaa43 can be Asn; and Xaa45 can be Phe. In yetanother embodiment, 15 or more of the amino acids are defined asfollows: Xaa10 can be Asp; Xaa11 can be Asp; Xaa13 can be Pro; Xaa15 canbe Arg; Xaa16 can be Ala; Xaa17 can be Ala; Xaa18 can be His; Xaa19 canbe Pro; Xaa21 can be Trp; Xaa22 can be Phe; Xaa23 can be Phe; Xaa31 canbe Glu; Xaa32 can be Glu; Xaa34 can be Ile; Xaa35 can be Tyr; Xaa39 canbe Glu; Xaa40 can be Gly; Xaa43 can be Asn; and Xaa45 can be Phe.

[0013] In a particular embodiment, the invention is directed to acomposition comprising a polypeptide as defined by SEQ ID NO:1, suchthat, if present, Xaa3 is Ser, Xaa2 is His, Xaa1 is Met, Xaa56 is Thr,Xaa57 is Arg, and/or Xaa58 is Asp, and methods of using suchcompositions.

[0014] In another embodiment, the invention is directed to a compositioncomprising a polypeptide comprising the amino acid sequence:

[0015] Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg AlaAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe IleTyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp (SEQ ID NO:2), and methods of using suchcompositions.

[0016] In a particular embodiment, the present invention is directed toa composition comprising a kallikrein binding polypeptide of 53-60 aminoacids comprising a Kunitz domain, wherein the Kunitz domain comprisesthe potential for disulfide bonds between cysteines at positions 5 and55; 14 and 38; and 30 and 51 (according to amino acid positionscorresponding to bovine pancreatic trypsin inhibitor (BPTI)), andfurther comprising:

[0017] amino acid number 13 selected from His and Pro;

[0018] amino acid number 16 selected from Ala and Gly;

[0019] amino acid number 17 selected from Ala, Asn, and Ser;

[0020] amino acid number 18 selected from His and Leu; and

[0021] amino acid number 19 selected from Gln, Leu, and Pro (SEQ IDNO:23).

[0022] In a particular embodiment, the present invention is directed toa composition comprising a kallikrein binding polypeptide of 53-60 aminoacids comprising a Kunitz domain, wherein the Kunitz domain comprisesthe potential for disulfide bonds between cysteines at positions 5 and55; 14 and 38; and 30 and 51 (according to amino acid positionscorresponding to bovine pancreatic trypsin inhibitor (BPTI)), andfurther comprising:

[0023] amino acid number 13 selected from His and Pro;

[0024] amino acid number 15 selected from Lys and Arg;

[0025] amino acid number 16 selected from Ala and Gly;

[0026] amino acid number 17 selected from Ala, Asn, and Ser;

[0027] amino acid number 18 selected from His and Leu; and

[0028] amino acid number 19 selected from Gln, Leu, and Pro,

[0029] amino acid number 31 is Glu;

[0030] amino acid number 32 selected from Glu and Gln;

[0031] amino acid number 34 selected from Ser, Thr, and Ile; and

[0032] amino acid number 39 selected from Gly, Glu, and Ala (SEQ IDNO:24).

[0033] In a particular embodiment, the present invention is directed toa composition comprising a kallikrein binding polypeptide of 53-60 aminoacids comprising a Kunitz domain, wherein the Kunitz domain comprises acysteine at each of positions 5 and 55; 14 and 38; and 30 and 51(according to amino acid positions corresponding to bovine pancreatictrypsin inhibitor (BPTI)), and further comprising:

[0034] amino acid number 13 selected from His and Pro;

[0035] amino acid number 15 selected from Lys and Arg;

[0036] amino acid number 16 selected from Ala and Gly;

[0037] amino acid number 17 selected from Ala, Asn, and Ser;

[0038] amino acid number 18 selected from His and Leu; and

[0039] amino acid number 19 selected from Gln, Leu, and Pro,

[0040] amino acid number 31 is Glu;

[0041] amino acid number 32 selected from Glu and Gln;

[0042] amino acid number 34 selected from Ser, Thr, and Ile; and

[0043] amino acid number 39 selected from Gly, Glu, and Ala (SEQ IDNO:24).

[0044] In a particular embodiment, the Kunitz domain is selected fromthe group consisting of:

[0045] KKII/3 #1 (SEQ ID NO:24)

[0046] KKII/3 #2 (SEQ ID NO:25)

[0047] KKII/3 #3 (SEQ ID NO:26)

[0048] KKII/3 #4 (SEQ ID NO:27)

[0049] KKII/3 #5 (SEQ ID NO:28)

[0050] KKII/3 #6 (SEQ ID NO:29)

[0051] KKII/3 #7 (SEQ ID NO:30)

[0052] KKII/3 #8 (SEQ ID NO:31)

[0053] KKII/3 #9 (SEQ ID NO:32) and

[0054] KKII/3 #10 (SEQ ID NO:33) as described in Table 1.

[0055] Each of the compositions described herein can be used in themethods of the invention. Further, the compounds described herein can beused in the manufacture of a medicament or composition for theindications or methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 is a simplified diagram of major multiple pathways andrelated events involved in the contact activation system and systemicinflammatory response (SIR) that may arise in a patient subjected tosoft and bone tissue trauma such as that associated with a coronaryartery bypass grafting (CABG) procedure, especially when the CABGprocedure involves extra-corporeal blood circulation, such ascardiopulmonary bypass (CPB; Bypass Apparatus). Arrows indicateactivation from one component or event to another component or event inthe cascade. Arrows in both directions indicate activating effects ofcomponents or events in both directions. Broken arrows indicate likelyparticipation of one component or event in the activation of anothercomponent or event. Abbreviations are as follows: “tPA”=tissueplasminogen activator; “C5a”=a protein component of the complementsystem; “fxIIa”=activator protein of pre-kallikrein to form activekallikrein; “Extrinsic”=extrinsic coagulation system;“Intrinsic”=intrinsic coagulation system.

[0057]FIG. 2 shows a portion of a DNA and corresponding deduced aminoacid for a kallikrein inhibitor (“KI”) polypeptide of the invention inplasmid pPIC-K503. The inserted DNA encodes the mata prepro signalpeptide of Saccharomyces cerevisiae (underlined) fused in frame to theamino terminus of the PEP-1 KI polypeptide having the amino acidsequence enclosed by the boxed area. The amino acid sequence of thePEP-1 KI polypeptide shown in the boxed region is SEQ ID NO:2, and thecorresponding nucleotide coding sequence of the KI polypeptide is SEQ IDNO:3. The dashed arrows indicate the location and direction of two PCRprimer sequences in AOX regions that were used to produce sequencingtemplates. DNA sequence for the entire nucleotide sequence of the figurecomprises the structural coding sequence for the fusion protein and isdesignated SEQ ID NO:35. The double underlined portion of the sequenceindicates a diagnostic probe sequence. BstBI and EcoRI indicatelocations of their respective palindromic, hexameric, restrictionendonuclease sites in the sequence. Asterisks denote translational stopcodons.

[0058]FIG. 3 shows an alignment of amino acid sequences of the preferredembodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0059] A description of preferred embodiments of the invention follows.

[0060] The invention is based on the discovery of kallikrein inhibitor(KI) polypeptides that inhibit plasma kallikrein with a specificity thatpermits their use in improved methods of preserving organs and tissues,such as pending a transplantation, and and to corresponding methods. Theinvention also relates to reducing, inhibiting or preventing reperfusioninjury or damage in an organ or tissue that has been removed from itshost and compositions therefor.

[0061] Polypeptides Useful in the Invention

[0062] KI polypeptides useful in the invention comprise Kunitz domainpolypeptides. In one embodiment these Kunitz domains are variant formscomprising the looped structure of Kunitz domain 1 of humanlipoprotein-associated coagulation inhibitor (LACI) protein. LACIcontains three internal, well-defined, peptide loop structures that areparadigm Kunitz domains (Girard, T. et al., 1989. Nature, 338:518-520).The three Kunitz domains of LACI confer the ability to bind and inhibitkallikrein, although not with exceptional affinity. Variants of Kunitzdomain 1 of LACI described herein have been screened, isolated and bindkallikrein with enhanced affinity and specificity (see, for example,U.S. Pat. Nos. 5,795,865 and 6,057,287, incorporated herein byreference). An example of a preferred polypeptide useful in theinvention has the amino acid sequence defined by amino acids 3-60 of SEQID NO:2.

[0063] Kallikrein binding polypeptides can be used to target therapeuticor diagnostic molecules to kallikrein in, for example, organ tissue,cells, or whole organisms. Such methods of targeted delivery fortherapeutic or diagnostic purposes would be known to one of skill in theart. For example, targeted kallikrein binding polypeptides could be usedby one of skill in the art to identify an organ that has been damaged bythe effects of kallikrien, or kallikrein can be targeted for the effectsof a particular therapeutic agent using kallikrein binding polypeptidesof the invention.

[0064] Every polypeptide useful in the invention binds kallikrein. Inpreferred embodiments, the polypeptides are kallikrein inhibitors (KI)as determined using kallikrein binding and inhibition assays known inthe art. The enhanced affinity and specificity for kallikrein of thevariant Kunitz domain polypeptides described herein provides the basisfor their use in CPB and especially CABG surgical procedures to preventor reduce perioperative blood loss and/or SIR in patients undergoingsuch procedures. The KI polypeptides used in the invention can have orcomprise the amino acid sequence of a variant Kunitz domain polypeptideoriginally isolated by screening phage display libraries for the abilityto bind kallikrein.

[0065] KI polypeptides useful in the methods and compositions of theinvention comprise a Kunitz domain polypeptide comprising the amino acidsequence: (SEQ ID NO:1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 PheXaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58

[0066] “Xaa” refers to a position in a peptide chain that can be any ofa number of different amino acids. For example, for the KI peptidesdescribed herein, Xaa10 can be Asp or Glu; Xaa11 can be Asp, Gly, Ser,Val, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg, His, Asn, Ser, Thr,Ala, Gly, Lys or Gln; Xaa15 can be Arg, Lys, Ala, Ser, Gly, Met, Asn orGln; Xaa16 can be Ala, Gly, Ser, Asp or Asn; Xaa17 can be Ala, Asn, Ser,Ile, Gly, Val, Gln or Thr; Xaa18 can be His, Leu, Gln or Ala; Xaa19 canbe Pro, Gln, Leu, Asn or Ile; Xaa21 can be Trp, Phe, Tyr, His or Ile;Xaa31 can be Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile or Thr; Xaa32can be Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 canbe Ile, Thr, Ser, Val, Ala, Asn, Gly or Leu; Xaa35 can be Tyr, Trp orPhe; Xaa39 can be Glu, Gly, Ala, Ser or Asp. Amino acids Xaa6, Xaa7,Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41,Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54can be any amino acid. Additionally, each of the first four and at lastthree amino acids of SEQ ID NO:1 can optionally be present or absent andcan be any amino acid, if present.

[0067] Peptides defined according to SEQ ID NO:1 form a set ofpolypeptides that bind to and inhibit kallikrein. The diversity of theKI's is increased as the number of variable positions in the peptidesequence is increased or as the number of amino acids possible at avariable position increases. For example, in a preferred embodiment ofthe invention, a KI polypeptide useful in the methods and compositionsof the invention has the following variable positions: Xaa11 can be Asp,Gly, Ser or Val; Xaa13 can be Pro, Arg, His or Asn; Xaa15 can be Arg orLys; Xaa16 can be Ala or Gly; Xaa17 can be Ala, Asn, Ser or Ile; Xaa18can be His, Leu or Gln; Xaa19 can be Pro, Gln or Leu; Xaa21 can be Trpor Phe; Xaa31 is Glu; Xaa32 can be Glu or Gln; Xaa34 can be Ile, Thr orSer; Xaa35 is Tyr; and Xaa39 can be Glu, Gly or Ala.

[0068] A more specific embodiment of the claimed invention is defined bythe following amino acids at variable positions: Xaa10 is Asp; Xaa11 isAsp; Xaa13 can be Pro or Arg; Xaa15 is Arg; Xaa16 can be Ala or Gly;Xaa17 is Ala; Xaa18 is His; Xaa19 is Pro; Xaa21 is Trp; Xaa31 is Glu;Xaa32 is Glu; Xaa34 can be Ile or Ser; Xaa35 is Tyr; and Xaa39 is Gly.

[0069] Also encompassed within the scope of the invention are peptidesthat comprise portions of the polypeptides described herein. Forexample, polypeptides could comprise binding domains for specifickallikrein epitopes. Such fragments of the polypeptides described hereinwould also be encompassed.

[0070] KI polypeptides useful in the methods and compositions describedherein comprise a Kunitz domain. A subset of the sequences encompassedby SEQ ID NO:1 are described by the following (where not indicated,“Xaa” refers to the same set of amino acids that are allowed for SEQ IDNO:1): (SEQ ID NO:36) Met His Ser Phe Cys Ala Phe Lys Ala Xaa10 Xaa11Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21 Phe Phe Asn IlePhe Thr Arg Gln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.

[0071] Specific and particular examples of KI peptides useful in theinvention described herein are as follows: Met His Ser Phe Cys Ala PheLys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg SEQ ID NO:2) Trp PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp(amino acids 3-60 of, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyPro Cys Lys Ala Asn His Leu Arg (SEQ ID NO:4) Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg (SEQ IDNO:5) Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Thr Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysLys Ala Asn His Gln Arg (SEQ ID NO:6) Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Gln Phe Thr Tyr Gly Gly Cys Ala Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Ala Ser Leu Pro Arg (SEQ ID NO:7)Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys AlaAsn His Gln Arg (SEQ ID NO:8) Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly His Cys Lys Gly Ala His Leu Arg (SEQ ID NO:9) Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Lys Gly Ala HisLeu Arg (SEQ ID NO:10) Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala AspGly Gly Arg Cys Arg Gly Ala His Pro Arg (SEQ ID NO:11) Trp Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg(SEQ ID NO:12) Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Val GlyArg Cys Arg Gly Ala His Pro Arg (SEQ ID NO:13) Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Gln Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser PheCys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala Gln Pro Arg (SEQ IDNO:14) Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Gln Gln Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Gln Gln Cys Lys Lys MetCys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Ser CysArg Ala Ala His Leu Arg (SEQ ID NO:15) Trp Phe Phe Asn Ile Phe Thr ArgGln Cys Gln Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GlnSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys AlaPhe Lys Ala Glu Gly Gly Ser Cys Arg Ala Ala His Gln Arg (SEQ ID NO:16)Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Gln Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg GlyAla His Leu Arg (SEQ ID NO:17) Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly His Cys Arg Gly Ala Leu Pro Arg (SEQ ID NO:18) Trp PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Asn Cys Arg Gly Asn LeuPro Arg (SEQ ID NO:19) Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala AspSer Gly Arg Cys Arg Gly Asn His Gln Arg (SEQ ID NO:20) Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met HisSer Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg Ala Ile Gln Pro Arg(SEQ ID NO:21) Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyArg Cys Arg Gly Ala His Pro Arg (SEQ ID NO:22) Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.

[0072]FIG. 3 provides an amino acid sequence alignment of thesesequences.

[0073] Other KI polypeptides useful in the present invention include:Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala Arg IleIle Arg (SEQ ID NO:23) Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln ThrPhe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala Glu AspCys Met Arg Thr Cys Gly Gly Ala, Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly His Cys Lys Ala Ser Leu Pro Arg (SEQ ID NO:24) Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Asn His Leu Arg(SEQ ID NO:25) Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyHis Cys Lys Ala Asn His Gln Arg (SEQ ID NO:26) Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg (SEQ IDNO:27) Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln Phe Thr Tyr GlyGly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysLys Ala Ser Leu Pro Arg (SEQ ID NO:28) Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg (SEQ ID NO:29)Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys AlaAsn His Gln Arg (SEQ ID NO:30) Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Gln Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly His Cys Lys Ala Asn His Gln Arg (SEQ ID NO:31) Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Gln Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn HisGln Arg (SEQ ID NO:32) Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly His Cys Lys Gly Ala His Leu Arg (SEQ ID NO:33) Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Ile Met Lys Arg(SEQ ID NO:34) Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe IleTyr Gly Gly Cys Gln Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp.

[0074] These sequences are summarized in the following Table 1. TABLE 1Amino acid sequences of LACI(K1) variants selected for binding to humanplasma kallikrein. 13 16 17 18 19 31 32 34 39 (a) KKII/3#1 H A S L P E EI E (SEQ ID NO:24) KKII/3#2 P A N H L E E S G (SEQ ID NO:25) KKII/3#3 HA N H Q E E T G (SEQ ID NO:26) KKII/3#4 H A N H Q E Q T A (SEQ ID NO:27)KKII/3#5 H A S L P E E I G (SEQ ID NO:28) KKII/3#6 H A N H Q E E S G(SEQ ID NO:29) KKII/3#7 H A N H Q E E S G (SEQ ID NO:30) KKII/3#8 H A NH Q E E S G (SEQ ID NO:31) KKII/3#9 H A N H Q E E S G (SEQ ID NO:32)KKII/3#10 H G A H L E E I E (SEQ ID NO:33) Consensus H A N H Q E E S/T G

[0075] The polypeptides useful in the methods and compositions describedherein may be made synthetically using any standard polypeptidesynthesis protocol and equipment. For example, the stepwise synthesis ofa KI polypeptide described herein may be carried out by the removal ofan amino (N) terminal-protecting group from an initial (i.e.,carboxy-terminal) amino acid, and coupling thereto of the carboxyl endof the next amino acid in the sequence of the polypeptide. This aminoacid is also suitably protected. The carboxyl group of the incomingamino acid can be activated to react with the N-terminus of the boundamino acid by formation into a reactive group such as formation into acarbodiimide, a symmetric acid anhydride, or an “active ester” groupsuch as hydroxybenzotriazole or pentafluorophenyl esters. Preferredsolid-phase peptide synthesis methods include the BOC method, whichutilizes tert-butyloxycarbonyl as the a-amino protecting group, and theFMOC method, which utilizes 9-fluorenylmethloxycarbonyl to protect thea-amino of the amino acid residues. Both methods are well known to thoseof skill in the art (Stewart, J. and Young, J., Solid-Phase PeptideSynthesis (W. H. Freeman Co., San Francisco 1989); Merrifield, J., 1963.Am. Chem. Soc., 85:2149-2154; Bodanszky, M. and Bodanszky, A., ThePractice of Peptide Synthesis (Springer-Verlag, New York 1984), theentire teachings of these references is incorporated herein byreference). If desired, additional amino- and/or carboxy-terminal aminoacids may be designed into the amino acid sequence and added duringpolypeptide synthesis.

[0076] Alternatively, Kunitz domain polypeptides and KI polypeptidesuseful in the compositions and methods of the invention may be producedby recombinant methods using any of a number of cells and correspondingexpression vectors, including but not limited to bacterial expressionvectors, yeast expression vectors, baculovirus expression vectors,mammalian viral expression vectors, and the like. Kunitz domainpolypeptides and KI polypeptides useful in the compositions and methodsof the invention may also be produced transgenically using nucleic acidmolecules comprising a coding sequence for a Kunitz domain or KIpolypeptide described herein, wherein the nucleic acid molecule can beintegrated into and expressed from the genome of a host animal usingtransgenic methods available in the art. In some cases, it may benecessary or advantageous to fuse the coding sequence for a Kunitzdomain polypeptide or a KI polypeptide comprising the Kunitz domain toanother coding sequence in an expression vector to form a fusionpolypeptide that is readily expressed in a host cell. Preferably, thehost cell that expresses such a fusion polypeptide also processes thefusion polypeptide to yield a Kunitz domain or KI polypeptide useful inthe invention that contains only the desired amino acid sequence.Obviously, if any other amino acid(s) remain attached to the expressedKunitz domain or KI polypeptide, such additional amino acid(s) shouldnot diminish the kallikrein binding and/or kallikrein inhibitoryactivity of the Kunitz domain or KI polypeptide so as to preclude use ofthe polypeptide in the methods or compositions of the invention.

[0077] A preferred recombinant expression system for producing KIpolypeptides useful in the methods and compositions described herein isa yeast expression vector, which permits a nucleic acid sequenceencoding the amino acid sequence for a KI polypeptide or Kunitz domainpolypeptide to be linked in the same reading frame with a nucleotidesequence encoding the mata prepro leader peptide sequence ofSaccharomyces cerevisiae, which in turn is under the control of anoperable yeast promoter. The resulting recombinant yeast expressionplasmid may then be transformed by standard methods into the cells of anappropriate, compatible yeast host, which cells are able to express therecombinant protein from the recombinant yeast expression vector.Preferably, a host yeast cell transformed with such a recombinantexpression vector is also able to process the fusion protein to providean active KI polypeptide useful in the methods and compositions of theinvention. A preferred yeast host for producing recombinant Kunitzdomain polypeptides and KI polypeptides comprising such Kunitz domainsis Pichia pastoris.

[0078] As noted above, KI polypeptides that are useful in the methodsand compositions described herein may comprise a Kunitz domainpolypeptide described herein. Some KI polypeptides may have anadditional flanking sequence, preferably of one to six amino acids inlength, at the amino and/or carboxy-terminal end, provided suchadditional amino acids do not significantly diminish kallikrein bindingaffinity or kallikrein inhibition activity so as to preclude use in themethods and compositions described herein. Such additional amino acidsmay be deliberately added to express a KI polypeptide in a particularrecombinant host cell or may be added to provide an additional function,e.g., to provide a peptide to link the KI polypeptide to anothermolecule or to provide an affinity moiety that facilitates purificationof the polypeptide. Preferably, the additional amino acid(s) do notinclude cysteine, which could interfere with the disulfide bonds of theKunitz domain. Native examples of Kunitz domains exhibit disulfidebonds, e.g., BPTI contains disulfide bonds between cysteine residues atamino acid positions 5 and 55; 14 and 38; and 30 and 51

[0079] An example of a preferred Kunitz domain polypeptide useful in themethods and compositions of the invention has the amino acid sequence ofresidues 3-60 of SEQ ID NO:2. When expressed and processed in a yeastfusion protein expression system (e.g., based on the integratingexpression plasmid pHIL-D2), such a Kunitz domain polypeptide retains anadditional amino terminal Glu-Ala dipeptide from the fusion with themata prepro leader peptide sequence of S. cerevisiae. When secreted fromthe yeast host cell, most of the leader peptide is processed from thefusion protein to yield a functional KI polypeptide (also referred to as“PEP-1” or “DX88”) having the amino acid sequence of SEQ ID NO:2 (seeboxed region in FIG. 2).

[0080] Particularly preferred KI polypeptides useful in the methods andcompositions described herein have a binding affinity for kallikreinthat is on the order of 1000 times higher than that of aprotinin, whichis currently approved for use in CABG procedures to reduce blood loss.The surprisingly high binding affinities of such KI polypeptidesdescribed herein indicate that such KI polypeptides exhibit a highdegree of specificity for kallikrein to the exclusion of other moleculartargets (see Table 1, below). Thus, use of such polypeptides accordingto the invention reduces much of the speculation as to the possibletherapeutic targets. The lower degree of specificity exhibited by, forexample, aprotinin, leads to possible pleiotropic side effects andambiguity as to its therapeutic mechanism.

[0081] The polypeptides defined by, for example, SEQ ID NO:1 containinvariant positions, e.g., positions 5, 14, 30, 51 and 55 can be Cysonly. Other positions such as, for example, positions 6, 7, 8, 9, 20,24, 25, 26, 27, 28, 29, 41, 42, 44, 46, 47, 48, 49, 50, 52, 53 and 54can be any amino acid (including non-naturally occurring amino acids).In a particularly preferred embodiment, one or more amino acidscorrespond to that of a native sequence (e.g., LACI (SEQ ID NOS:32-34)).In a preferred embodiment, at least one variable position is differentfrom that of the native sequence. In yet another preferred embodiment,the amino acids can each be individually or collectively substituted bya conservative or non-conservative amino acid substitution. Conservativeamino acid substitutions replace an amino acid with another amino acidof similar chemical structure and may have no affect on proteinfunction. Non-conservative amino acid substitutions replace an aminoacid with another amino acid of dissimilar chemical structure. Examplesof conserved amino acid substitutions include, for example, Asn-<Asp,Arg-<Lys and Ser-<Thr. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 and/or 21 of these aminoacids can be independently or collectively, in any combination, selectedto correspond to the corresponding position of SEQ ID NO:2.

[0082] Other positions, for example, positions 10, 11, 13, 15, 16, 17,18, 19, 21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, can be any of aselected set of amino acids. Thus SEQ ID NO:1 defines a set of possiblesequences. Each member of this set contains, for example, a cysteine atpositions 5, 14, 30, 51 and 55, and any one of a specific set of aminoacids at positions 10, 11, 13, 15, 16, 17, 18, 19, 221, 22, 23, 31, 32,34, 35, 39, 40, 43 and 45. In a preferred embodiment, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and/or 19 of these aminoacids can be independently or collectively, in any combination, selectedto correspond to the corresponding position of SEQ ID NO:2. The peptidepreferably has at least 80%, at least 85%, at least 90% or at least 95%identity to SEQ ID NO:2.

[0083] Methods and Compositions

[0084] The present invention is directed to methods for preservingorgans and tissues comprising contacting the organ or tissue with apreservative solution comprising a kallikrein inhibitor, such as thosedescribed herein. The invention also relates to reducing, inhibiting orpreventing reperfusion injury or damage in an organ or tissue that hasbeen removed from its host comprising contacting the organ or tissuewith a kallikrein inhibitor. The preservative solutions of the inventioncan be used to preserve and/or protect organ tissue, or whole organs,when said organs or tissue are brought into contact with the solution. Aspecific embodiment of the invention is for the preservation of a humanheart, or human myocardial tissue. Another embodiment of the inventionis for the preservation of a human lung or human lung tissue. Otherorgans, or parts thereof, that can be preserved according to theinvention include kidney, liver, endothelial tissue, intestinal tissue,vascular tissue (e.g., an aorta graft), skin, and pancreas. Theinvention contemplates the use of the solutions to preserve mammaliantissue, organs or portion thereof. In addition, the solutions can beused to facilitate transplantation of organs, e.g., by perfusion of theorgan or tissue during the transplantation procedure. The solution canalso be used as a cardioplegia solution in cardiac surgery. Preferably,the organ or portion thereof, is maintained in the appropriate solutionat all times, particularly prior to the transplant procedure.

[0085] The solutions of the invention can be used to maintain viabilityof the organ or tissue during storage, transplantation or other surgery.The invention includes a method of storing tissue or organs comprisingcontacting said tissue, organ or part thereof, with the solution of theinvention, such that the in vivo and/or in vitro viability is prolonged.The solutions permit maintenance of viability of heart or lung tissuefor up to 24 hours or more. Use of the solutions of the inventionresults in improved organ viability.

[0086] Alternatively or in addition, once removed from the donor, theorgan or living tissue may be placed in a preservation solutioncontaining the inhibitor. In addition, the kallikrein inhibitor is alsopreferably administered to the transplant recipient just prior to, orconcommitant with, transplantation. In all cases, the inhibitor also canbe administered directly to the tissue at risk, as by injection to thetissue, or it may be provided systemically, either by oral or parenteraladministration, using any of the methods and formulations describedherein and/or known in the art.

[0087] In one embodiment, any commercially available preservationsolution may be used to advantage. Examples of such solutions includethe Belzer UW solution sold under the trademark VIASPAN, described inU.S. Pat. Nos. 4,798,824, 4,873,230, 4,879,283, which are herebyincorporated by reference.

[0088] The preservation solution and perfusate composition described inthe aforementioned patents includes, but is not limited to, thefollowing: TABLE 2 Substance Amount in 1 Liter K⁺ - lactobionate 100mmol KH₂ PO₄ 25 mmol MgSO₄ 5 mmol Raffinose 30 mmol Adenosine 5 mmolGlutathione 3 mmol Insulin 100 U Bactrim 0.5 mL Dexamethasone 8 mgAllopurinol 1 mM Hydroxyethyl starch having a 50 g molecular weight ofabout 200,000 to about 300,000 daltons and a degree of substitution offrom about 0.4 to 0.7

[0089] The solution is brought to pH 7.4 at room temperature with NaOH.The final concentrations are Na=30.±0.5 mM, K⁺=120±5 mM,mOsm/liter=320±5. Bactrim=trimethoprim (16 mg/mL) and sulfamethoxazole(80 mg/mL). The hydroxyethyl starch can be present in the range of fromabout 3 to about 8%.

[0090] This solution typically provides for a 72 hour preservation ofthe pancreas, 48 hour preservation for the kidney and at least 24 hourpreservation for the liver. U.S. Pat. No. 5,145,771, incorporated hereinby reference, described the organ preservation solution known as the“Carolina Solution,” which is also useful in the present invention. Therinse or preservation solution composition described in theaforementioned patent includes, but is not limited to, the components inabout the concentration ranges set forth in Table 3 below. TABLE 3Concentration Ranges in 1 Liter 10% modified hydroxyethyl starch 30 g/Lto 100 g/L NaCl 85 mM to 145 mM KCl 3 mM to 6 mM CaCl₂ 1.0 mM to 1.6 mMKH₂ PO₄ 0.7 mM to 1.3 mM MgSO₄ 0.9 mM to 1.5 mM Allopurinol 0.05 mM to5.0 mM Desferrioxamine 0.02 mM to 2.0 mM Glutathione 0.5 mM to 10.0 mMNicardipene 0.1 .mu.M to 5.0 .mu.M Adenosine 0.1 mM to 5.0 mM Fructose1.0 mM to 50.0 mM Glucose 1.0 mM to 50.0 mM Insulin 5 U/L to 250 U/LMops 2 mM 40 mM

[0091] One specific embodiment is prepared with the components in theamounts set forth in Table 4 below in accordance with the instructionsset forth below. TABLE 4 Components of 1 Liter Rinse Solution 500 mLDistilled Deionized Water 50 g/L 10% modified hydroxyethyl starch 115 mMNaCl 6.7 g 5 mM KCl 0.37 g 1.30 mM CaCl₂ 0.19 g 1 mM KH₂ PO₄ 0.14 g 1.2mM MgSO₄ 0.15 g 1 mM Allopurinol 0.14 g 1 mM Desferrioxamine 0.65 g 3 mMGlutathione 0.92 g 2 .mu.M Nicardipene 0.80 mg 1 mM Adenosine 0.32 g 10mM Fructose 1.8 g 10 mM Glucose 1.8 g 100 U/L Insulin 100 units 20 mMMops 4.2 g

[0092] In one embodiment this solution can be prepared as follows: usinga 500 mL volumetric flask, measure 500 mL of 10% (weight/volume)hydroxyethyl starch solution and pour into a 1 L beaker. Add 400 mL ofdouble distilled water and stir vigorously using a magnetic stir bar.Add the rest of the components one at a time. After all components areadded, adjust the pH to 6.5 with 1-2 mL 5N NaOH. The solution should bestirred for at least thirty minutes. Transfer the solution to a 1 Lvolumetric flask and bring to 1 L final volume. Filter to remove anyundissolved particles.

[0093] Still another embodiment is exemplified by Table 5 below. TABLE 5Concentration Ranges in 1 Liter NaCl 85 mM to 145 mM KCl 3 mM to 6 mMCaCl₂ 1.0 mM to 1.6 mM KH₂ PO₄ 0.7 mM to 1.3 mM MgSO₄ 0.9 mM to 1.5 mMAdenosine 0.12 mM to 1.2 mM

[0094] A composition according to Table 5 above may optionally includeone, several, or all of the further ingredients specified in Table 3above. Preferably, the composition includes at least one antioxidant.Thus, one specific embodiment of a composition is set forth in Table 6below: TABLE 6 Components of 1 Liter Rinse Solution 500 mL DistilledDeionized Water 115 mM NaCl 6.7 g 5 mM KCl 0.37 g 1.30 mM CaCl₂ 0.19 g 1mM KH₂PO₄ 0.14 g 1.2 mM MgSO₄ 0.15 g 1 mM Allopurinol 0.14 g 1 mMDesferrioxamine 0.65 g 3 mM Glutathione 0.92 g .12 mM Adenosine 0.038 g

[0095] Preferred compositions may further comprise one or morepharmaceutically acceptable buffers, carriers, antioxidants, proteaseinhibitors, or other anti-ischemia agents.

[0096] Compositions useful in the methods of the invention comprise anyof the Kunitz domain polypeptides or KI polypeptides comprising suchKunitz domain polypeptides described herein. Particularly preferred areKI polypeptides comprising a Kunitz domain polypeptide having a 58-aminoacid sequence of amino acids 3-60 of SEQ ID NO:2. An example of such aparticularly preferred KI polypeptide useful in the methods andcompositions of the invention is the PEP-1 KI polypeptide having the60-amino acid sequence of SEQ ID NO:2. A nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:2 is provided in SEQ ID NO:3 (see,e.g., nucleotides 309-488 in FIG. 2). It is understood that based on theknown genetic code, the invention also provides degenerate forms of thenucleotide sequence of SEQ ID NO:3 by simply substituting one or more ofthe known degenerate codons for each amino acid encoded by thenucleotide sequence. Nucleotides 7-180 of SEQ ID NO:3, and degenerateforms thereof, encode the non-naturally occurring Kunitz domainpolypeptide having the 58-amino acid sequence of amino acids 3-60 of SEQID NO:2.

[0097] Concentration Considerations for KI Polypeptides

[0098] Several considerations regarding dosing with a KI polypeptide inmethods of the invention may be illustrated by way of example with therepresentative PEP-1 KI polypeptide of the invention having the aminosequence of SEQ ID NO:2 (molecular weight of 7,054 Daltons).

[0099] Table 7, below, provides a comparison of the affinity (K_(i,app))of the PEP-1 KI polypeptide for kallikrein and eleven other known plasmaproteases. TABLE 7 Protease Substrate PEP-1 K_(i,app) (pM) AprotininK_(i,app (pM)) human plasma kallikrein 44 3.0 × 10⁴ human urinekallikrein  >1 × 10⁸ 4.0 × 10³ porcine pancreatic kallikrein  2.7 × 10⁷550 human C1r, activated >2.0 × 10⁸ 1.0 × 10⁷ human C1s, activated >2.0× 10⁷ >1.0 × 10⁸  human plasma factor XIa  1.0 × 10⁴ ND human plasmafactor XIIa >2.0 × 10⁷ >1.0 × 10⁸  human plasmin  1.4 × 10⁵ 894 humanpancreatic trypsin  >2 × 10⁷ ND human pancreatic chymotrypsin >2.0 × 10⁷7.3 × 10⁵ human neutrophil elastase >2.0 × 10⁷ 1.7 × 10⁶ human plasmathrombin >2.0 × 10⁷ >1.0 × 10⁸ 

[0100] Clearly, the PEP-1 KI polypeptide is highly specific for humanplasma kallikrein. Furthermore, the affinity (K_(i,app)) of PEP-1 forkallikrein is 1000 times higher than the affinity of aprotinin forkallikrein: the K_(i,app) of PEP-1 for kallikrein is about 44 pM (Table1), whereas the K_(i,app) of aprotinin for kallikrein is 30,000 pM.Thus, a dose of PEP-1 could be approximately 1000 times lower than thatused for aprotinin on a per mole basis. However, consideration ofseveral other factors may provide a more accurate estimation of the doseof PEP-1 required in practice. Such factors include the amount ofkallikrein activated upon organ removal from a particular patient, andwill be recognized by the skilled artisan.

[0101] The invention will be further described with reference to thefollowing non-limiting examples. The teachings of all the patents,patent applications and all other publications and websites cited hereinare incorporated by reference in their entirety.

EXEMPLIFICATION Example 1

[0102] A Representative KI Polypeptide.

[0103] A KI polypeptide (PEP-1) useful in the compositions and methodsof the invention was identified as a kallikrein binding polypeptidedisplayed on a recombinant phage from a phage display library. PEP-1 hasthe following amino acid sequence: Glu Ala Met His Ser Phe Cys Ala PheLys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn IlePhe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln AsnArg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ IDNO:2). The molecular weight of PEP-1 is 7,054 Daltons.

[0104] The nucleotide sequence (SEQ ID NO:3) of the recombinant phageDNA encoding the PEP-1 amino acid sequence (amino acids 3-60 of SEQ IDNO:2) was isolated and sequenced by standard methods determined from therecombinant phage DNA. PEP-1 was produced in amounts useful for furthercharacterization as a recombinant protein in His4⁻ phenotype host cellsof yeast strain Pichia pastoris.

Example 2

[0105] Construction of a Recombinant Plasmid to Express KI Polypeptides.

[0106] The initial plasmid, pHIL-D2, is ampicillin resistant andcontains a wild-type allele of His4 from P. pastoris. The final DNAsequence comprising the coding sequence for the mata Prepro-PEP-1 fusionprotein in the recombinant expression plasmid pPIC-K503 is shown in FIG.2. The DNA sequence of pHIL-D2 was modified to produce pPIC-K503, asfollows:

[0107] 1. The BstBI site in the 3′ AOX1 region of pHIL-D2, locateddownstream of the His4 gene, was removed by partial restrictiondigestion, fill-in, and ligation, altering the sequence from TTCGAA (SEQID NO:23) to TTCGCGAA (SEQ ID NO:24). This modification was made tofacilitate and direct the cloning of the expression cassette into theplasmid.

[0108] 2. The AatII site bearing the bla gene located downstream of His4was removed by restriction digestion, fill-in, and ligation modifyingthe sequence from GACGTC (SEQ ID NO:25) to GACGTACGTC (SEQ ID NO:26).This modification was made to facilitate the cloning of expressioncassettes having AatII sites into the plasmid. The DNA encoding PEP-1was synthesized based on the nucleotide sequence from the originalkallikrein-binding display phage and consisted of 450 base pairs (bp).The final DNA sequence of the insert in the pHIL-D2 plasmid is flankedby a 5′ AOX1 sequence and a 3′ AOX1 sequence (portions of which areshown in FIG. 2) and encode a fusion protein comprising the mata preprosignal peptide of S. cerevisiae fused to the structural coding sequencefor the PEP-1 KI polypeptide. The signal peptide was added to facilitatethe secretion of PEP-1 from the yeast host cells. The oligonucleotidesto form the insert were synthesized and obtained commercially (GenesisLabs, The Woodlands, Tex.), and the insert was generated by polymerasechain reaction (PCR). The linked synthetic DNA encoding the mataprepro/PEP-1 fusion protein was then incorporated by ligation into themodified pHIL-D2 plasmid between the BstBI and EcoRI sites.

[0109] The ligation products were used to transform Escherichia colistrain XL1 Blue. A PCR assay was used to screen E. coli transformantsfor the desired plasmid construct. DNA from cell extracts was amplifiedby PCR using primers containing the 5′ AOX1 and 3′ AOX1 sequences (seeabove and FIG. 2). PCR products of the correct number of base pairs weresequenced. In addition, approximately 20-50 bp on either side of thecloning sites were sequenced, and the predicted sequence was obtained.The final DNA sequence of the insert in the pHIL-D2 plasmid (to yieldplasmid pPIC-K503) is shown in FIG. 2 along with portions of flanking 5′and 3′ AOX1 sequences and corresponding amino acid sequence of thefusion protein comprising the mata prepro signal peptide of S.cerevisiae fused to the structural coding sequence for the PEP-1 KIpolypeptide. A transformant with the desired expression plasmidconstruct, plasmid pPIC-K503, was selected for preparing yeast celllines for routine production of PEP-1.

Example 3

[0110] Manufacture of PEP-1 from Recombinant Yeast Cell Line.

[0111] Spheroplasts of P. pastoris GS115 having the His4⁻ phenotype weretransformed with the expression plasmid pPIC-K503 (above) followinglinearization of the plasmid at the SacI site and homologousrecombination of the plasmid DNA into the host 5′ AOX1 locus. Thephenotype of the production strain is His4⁺. The entire plasmid wasinserted into the 5′ AOX1 genomic sequence of the yeast.

[0112] Isolates from the transformation were screened for growth in theabsence of exogenous histidine with methanol as the sole carbon source.Greater than 95% of the transformants retained the wild-type ability togrow with methanol as the sole carbon source, thereby demonstrating thatthe plasmid had been inserted into the host genome by homologousrecombination rather than transplacement. These transformants did notrequire exogenous histidine for growth, thereby demonstrating that theplasmid had integrated into the host genome. Selected colonies werecloned. Small culture expression studies were performed to identifyclones secreting the highest levels of active PEP-1 into the culturemedium. PEP-1 secretion levels in clarified culture supernatantsolutions were quantified for PEP-1 levels by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and evaluated forkallikrein inhibition. A yeast clone was selected for PEP-1 productionbased on its high level of PEP-1 expression among cultures sampled.

[0113] Master and working cell banks of P. pastoris producing PEP-1 wereprepared commercially (MDS Pharma Services, Bothell, Wash.). A standardproduction of PEP-1 in yeast comprised three steps as follows: (1)preparation of the seed culture, (2) fermentation, and (3) recovery ofthe culture.

[0114] The seed culture step consisted of the inoculation of six flasks(300 mL each) containing sterile inoculum broth (yeast nitrogen base,potassium phosphate, and glycerol, pH=5) with the contents of a singlevial of a working cell bank of P. pastoris producing PEP-1. Flasks wereinoculated in an orbital shaker (300 rpm) for approximately 13 hours at30° C.±2° C.

[0115] Fermentations were performed in a closed 100 liter Braunfermenter filled with sterile broth. Each fermentation was initiatedwith the transfer of the contents of the six seed culture flasks to thefermenter. After approximately 24 hours, the glycerol in the fermenterbecame exhausted and additional glycerol was added for approximately 8additional hours.

[0116] A mixed feed phase, which lasted approximately 83 hours, was theninitiated by the addition of a glycerol and methanol feed. At the end ofthis time, the fermentation was terminated, and the fermenter contentswere diluted with purified water. The purification and processing ofPEP-1 consisted of five steps as follows: (1) expanded bedchromatography, (2) cation exchange chromatography, (3) hydrophobicinteraction chromatography (HIC), (4) ultrafiltration and diafiltration,and (5) final filtration and packaging.

[0117] The initial purification step consisted of expanded bedchromatography. The diluted fermenter culture was applied to theequilibrated column packed with Streamline SP resin (Amersham PharmaciaStreamline 200 chromatography column, Amersham Pharmacia, Piscataway,N.J.). The column was then washed (50 mM acetic acid, pH=3.0−3.5) in anup-flow mode to flush the yeast cells from the expanded bed. The topadaptor was raised above the expanded bed enhance washing. The flow wasstopped and the bed was allowed to settle. The adaptor was moved down sothat it was slightly above the settled bed. The direction of the flowwas reversed. The effluent was collected. Washing was continued in adownward mode using 50 mM sodium acetate, pH 4.0. The effluent wascollected. PEP-1 was eluted from the column using 50 mM sodium acetate,pH 6.0. The eluate was collected in a 50 liter container. The eluate wasthen filtered through a 0.22 m filter into a clean container located inthe purification site. Additional samples were collected for thedetermination of PEP-1 concentration. A cation exchange chromatographystep was then performed using the filtered eluate from the expanded bedcolumn. PEP-1 was eluted from the column using 15 mM trisodium citrate,pH 6.2.

[0118] Additional proteins were removed from the PEP-1 preparation byhydrophobic interaction chromatography (HIC). Prior to HIC, the eluatefrom the cation exchange column was diluted with ammonium sulfate. Theeluate was applied to the column, and the PEP-1 was eluted usingammonium sulfate (0.572 M) in potassium phosphate (100 mM), pH 7.0. Theeluate was collected in fractions based on A280 values. All fractionswere collected into sterile, pre-weighed PETG bottles.

[0119] Selected fractions were pooled into a clean container. The poolwas concentrated by ultrafiltration. The concentrated PEP-1 preparationwas immediately diafiltered against ten volumes of PBS, pH 7.0.

[0120] A final filtration step was performed prior to packaging in orderto minimize the bioburden in the bulk PEP-1. The bulk solution wasfiltered through a 0.22 m filter and collected into a sterile,pre-weighed PETG bottle. A sample was removed for lot release testing.The remainder of the bulk was dispensed aseptically into sterile PETGbottles and stored at −20° C.

Example 4

[0121] Kallikrein Inhibition Assay.

[0122] A kinetic test was used to measure inhibitory activity of KIpolypeptides, such as PEP-1. The kinetic assay measures fluorescencefollowing kallikrein-mediated cleavage of a substrate,prolylphenylalanylarginyl amino methyl coumarin. A known amount ofkallikrein was incubated with a serially diluted KI polypeptidereference standard or serially diluted KI polypeptide test samples, in asuitable reaction buffer on a microtiter plate. Each sample was run intriplicate. The substrate solution was added, and the plate readimmediately using an excitation wavelength of 360 nm and an emissionwavelength of 460 nm. At least two each of the reference standard andsample curves were required to have an R-squared value of 0.95 to beconsidered valid.

Example 5

[0123] Organ Preservation

[0124] HUVEC at confluence were washed in PBS and further incubated at 4degrees for 24-48 hours in a serum free medium (SFM). After coldstorage, cells were washed several times with PBS, and kallikrein (0.125U) and the specific kallikrein substrate S2302 were added to the cells.Changes in optical density were recorded. For light microscopyevaluation of cell-bound PEP-1, after cold storage, HUVEC were treatedwith PEP-1, formalin fixed and treated with rabbit anti-PEP-1 andperoxidase conjugated anti-rabbit IgG. The ability of HUVEC to producekallikrein was also evaluated on cell surface and in the supernatants ofcells maintained at 37° C. Kallikrein activity was 380±19 A.U. insupernatants of HUVEC maintained at 37° C.; no activity was measurableon the surface of the same cells. At light microscopy evaluation therewas significant binding of PEP-1 to the surface of HUVEC cold treatedfor 24 hours. The maximum of the binding was obtained by incubatingcells in presence of PEP-1 (5 mg/ml). Cell-bound PEP-1 retained theability to inhibit exogenous kallikrein. These results indicate thatPEP-1 binds to endothelial cells, maintaining its kallikrein inhibitoryactivity. Therefore it can be used to detect and modulate kinin-mediateddamage on the vascular surface.

[0125] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

1 41 1 58 PRT Artificial Sequence Polypeptide Inhibiting Kallikrein 1Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Cys Xaa Xaa 1 5 1015 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa 20 2530 Phe Xaa Xaa Gly Gly Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 4045 Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 50 55 2 60 PRT ArtificialSequence Isolated Binding Peptides 2 Glu Ala Met His Ser Phe Cys Ala PheLys Ala Asp Asp Gly Pro Cys 1 5 10 15 Arg Ala Ala His Pro Arg Trp PhePhe Asn Ile Phe Thr Arg Gln Cys 20 25 30 Glu Glu Phe Ile Tyr Gly Gly CysGlu Gly Asn Gln Asn Arg Phe Glu 35 40 45 Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp 50 55 60 3 179 DNA Artificial Sequence Coding Sequenceof Pep-1 3 gaggctatgc actctttctg tgctttcaag gctgacgacg gtcgtgcagagctgctcacc 60 caagatggtt cttcaacatc ttcacgcgtc aatgcgagga gttcatctacggtggttgtg 120 agggtaacca aaacagattc gagtctctag aggagtgtaa gaagatgtgtactagagac 179 4 58 PRT Artificial Sequence Isolated Binding Peptides 4Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala 1 5 1015 Asn His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 2530 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 4045 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 5 58 PRT ArtificialSequence Isolated Binding Peptides 5 Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly His Cys Lys Ala 1 5 10 15 Asn His Gln Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Thr Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys ThrArg Asp 50 55 6 58 PRT Artificial Sequence Isolated Binding Peptides 6Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 1015 Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 2530 Phe Thr Tyr Gly Gly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 4045 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 7 58 PRT ArtificialSequence Isolated Binding Peptides 7 Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly His Cys Lys Ala 1 5 10 15 Ser Leu Pro Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ile Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys ThrArg Asp 50 55 8 58 PRT Artificial Sequence Isolated Binding Peptides 8Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 1015 Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 2530 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 4045 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 9 58 PRT ArtificialSequence Isolated Binding Peptides 9 Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly His Cys Lys Gly 1 5 10 15 Ala His Leu Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ile Tyr Gly Gly Cys Glu GlyAsn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys ThrArg Asp 50 55 10 58 PRT Artificial Sequence Isolated Binding Peptides 10Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Lys Gly 1 5 1015 Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 2530 Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 4045 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 11 58 PRT ArtificialSequence Isolated Binding Peptides 11 Met His Ser Phe Cys Ala Phe LysAla Asp Gly Gly Arg Cys Arg Gly 1 5 10 15 Ala His Pro Arg Trp Phe PheAsn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys GlyGly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys Lys Met CysThr Arg Asp 50 55 12 58 PRT Artificial Sequence Isolated BindingPeptides 12 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys ArgAla 1 5 10 15 Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 13 58 PRTArtificial Sequence Isolated Binding Peptides 13 Met His Ser Phe Cys AlaPhe Lys Ala Asp Val Gly Arg Cys Arg Gly 1 5 10 15 Ala His Pro Arg TrpPhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 14 58 PRT Artificial Sequence Isolated BindingPeptides 14 Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg Cys ArgGly 1 5 10 15 Ala Gln Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 15 58 PRTArtificial Sequence Isolated Binding Peptides 15 Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Ser Cys Arg Ala 1 5 10 15 Ala His Leu Arg TrpPhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 16 58 PRT Artificial Sequence Isolated BindingPeptides 16 Met His Ser Phe Cys Ala Phe Lys Ala Glu Gly Gly Ser Cys ArgAla 1 5 10 15 Ala His Gln Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 17 58 PRTArtificial Sequence Isolated Binding Peptides 17 Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Pro Cys Arg Gly 1 5 10 15 Ala His Leu Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 18 58 PRT Artificial Sequence Isolated BindingPeptides 18 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys ArgGly 1 5 10 15 Ala Leu Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 19 58 PRTArtificial Sequence Isolated Binding Peptides 19 Met His Ser Phe Cys AlaPhe Lys Ala Asp Ser Gly Asn Cys Arg Gly 1 5 10 15 Asn Leu Pro Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 20 58 PRT Artificial Sequence Isolated BindingPeptides 20 Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Arg Cys ArgGly 1 5 10 15 Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 21 58 PRTArtificial Sequence Isolated Binding Peptides 21 Met His Ser Phe Cys AlaPhe Lys Ala Asp Gly Gly Arg Cys Arg Ala 1 5 10 15 Ile Gln Pro Arg TrpPhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 22 58 PRT Artificial Sequence Isolated BindingPeptides 22 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys ArgGly 1 5 10 15 Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 23 58 PRTArtificial Sequence Isolated Binding Peptides 23 Arg Pro Asp Phe Cys LeuGlu Pro Pro Tyr Thr Gly Pro Cys Lys Ala 1 5 10 15 Arg Ile Ile Arg TyrPhe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30 Phe Val Tyr Gly GlyCys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45 Glu Asp Cys Met ArgThr Cys Gly Gly Ala 50 55 24 58 PRT Artificial Sequence Isolated BindingPeptides 24 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys LysAla 1 5 10 15 Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 25 58 PRTArtificial Sequence Isolated Binding Peptides 25 Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Pro Cys Lys Ala 1 5 10 15 Asn His Leu Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 26 58 PRT Artificial Sequence Isolated BindingPeptides 26 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys LysAla 1 5 10 15 Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 27 58 PRTArtificial Sequence Isolated Binding Peptides 27 Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 10 15 Asn His Gln Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln 20 25 30 Phe Thr Tyr Gly GlyCys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 28 58 PRT Artificial Sequence Isolated BindingPeptides 28 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys LysAla 1 5 10 15 Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 29 58 PRTArtificial Sequence Isolated Binding Peptides 29 Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 10 15 Asn His Gln Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 30 58 PRT Artificial Sequence Isolated BindingPeptides 30 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys LysAla 1 5 10 15 Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 31 58 PRTArtificial Sequence Isolated Binding Peptides 31 Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Ala 1 5 10 15 Asn His Gln Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 32 58 PRT Artificial Sequence Isolated BindingPeptides 32 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys LysAla 1 5 10 15 Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 33 58 PRTArtificial Sequence Isolated Binding Peptides 33 Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Gly 1 5 10 15 Ala His Leu Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 20 25 30 Phe Ile Tyr Gly GlyCys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys LysMet Cys Thr Arg Asp 50 55 34 58 PRT Artificial Sequence Isolated BindingPeptides 34 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys LysAla 1 5 10 15 Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu 20 25 30 Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe GluSer Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 35 548DNA Artificial Sequence Coding sequence for fusion protein 35 cgacttttaacgacaacttg agaagatcaa aaaacaacta attattcgaa acg atg 56 Met 1 aga ttc ccatct atc ttc act gct gtt ttg ttc gct gct tcc tct gct 104 Arg Phe Pro SerIle Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala 5 10 15 ttg gct gct ccagtt aac acc act act gaa gac gag act gct caa att 152 Leu Ala Ala Pro ValAsn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile 20 25 30 cct gct gag gct gtcatc ggt tac tct gac ttg gaa ggt gac ttc gac 200 Pro Ala Glu Ala Val IleGly Tyr Ser Asp Leu Glu Gly Asp Phe Asp 35 40 45 gtc gct gtt ttg cca ttctct aac tct act aac aac ggt ttg ttg ttc 248 Val Ala Val Leu Pro Phe SerAsn Ser Thr Asn Asn Gly Leu Leu Phe 50 55 60 65 atc aac act acc atc gcttct atc gct gct aag gag gaa ggt gtt tcc 296 Ile Asn Thr Thr Ile Ala SerIle Ala Ala Lys Glu Glu Gly Val Ser 70 75 80 ctc gag aag aga gag gct atgcac tct ttc tgt gct ttc aag gct gac 344 Leu Glu Lys Arg Glu Ala Met HisSer Phe Cys Ala Phe Lys Ala Asp 85 90 95 gac ggt ccg tgc aga gct gct caccca aga tgg ttc ttc aac atc ttc 392 Asp Gly Pro Cys Arg Ala Ala His ProArg Trp Phe Phe Asn Ile Phe 100 105 110 acg cgt caa tgc gag gag ttc atctac ggt ggt tgt gag ggt aac caa 440 Thr Arg Gln Cys Glu Glu Phe Ile TyrGly Gly Cys Glu Gly Asn Gln 115 120 125 aac aga ttc gag tct cta gag gagtgt aag aag atg tgt act aga gac 488 Asn Arg Phe Glu Ser Leu Glu Glu CysLys Lys Met Cys Thr Arg Asp 130 135 140 145 tagtaagaat tcgccttagacatgactgtt cctcagttca agttgggcac ttacgagaag 548 36 58 PRT ArtificialSequence Isolated Binding Peptide 36 Met His Ser Phe Cys Ala Phe Lys AlaXaa Xaa Gly Xaa Cys Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Arg Xaa Phe Phe AsnIle Phe Thr Arg Gln Cys Xaa Xaa 20 25 30 Phe Xaa Xaa Gly Gly Cys Xaa GlyAsn Gln Asn Arg Phe Glu Ser Leu 35 40 45 Glu Glu Cys Lys Lys Met Cys ThrArg Asp 50 55 37 6 DNA Artificial Sequence Modified Cloning Site 37ttcgaa 6 38 8 DNA Artificial Sequence Modified Cloning Site 38 ttcgcgaa8 39 6 DNA Artificial Sequence Modified Cloning Site 39 gacgtc 6 40 10DNA Artificial Sequence Modified Cloning Site 40 gacgtacgtc 10 41 145PRT Artificial Sequence Fusion Protein 41 Met Arg Phe Pro Ser Ile PheThr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro ValAsn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala ValIle Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu ProPhe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr IleAla Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Glu Lys ArgGlu Ala Met His Ser Phe Cys Ala Phe Lys Ala 85 90 95 Asp Asp Gly Pro CysArg Ala Ala His Pro Arg Trp Phe Phe Asn Ile 100 105 110 Phe Thr Arg GlnCys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn 115 120 125 Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg 130 135 140 Asp 145

What is claimed is:
 1. A method for preserving an organ or tissuecomprising contacting the organ or tissue with an effective amount of akallikrein inhibitor.
 2. The method of claim 1, wherein the polypeptidecomprises a Kunitz domain.
 3. The method according to claim 2, whereinthe kallikrein inhibitor is a polypeptide comprising SEQ ID NO:2.
 4. Themethod according to claim 2, wherein the kallikrein inhibitor is apolypeptide comprising amino acids 3-60 of SEQ ID NO:2.
 5. The method ofclaim 1, wherein the organ or tissue is heart, lung, kidney, pancreas,liver, intestine, endothelial tissue, vascular tissue or skin.
 6. Amethod for preserving an organ or tissue comprising contacting the organor tissue with an effective amount of a kallikrein inhibitor comprisinga polypeptide comprising the amino acid sequence: Xaa1 Xaa2 Xaa3 Xaa4Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16 Xaa17Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41 Xaa42Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1), wherein Xaa1, Xaa2, Xaa3, Xaa4,Xaa56, Xaa57 or Xaa58 are each individually an amino acid or absent;Xaa6, Xaa7, Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29,Xaa41, Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 andXaa54 can be any amino acid; Xaa10 is an amino acid selected from thegroup consisting of: Asp and Glu; Xaa11 is an amino acid selected fromthe group consisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr;Xaa13 is an amino acid selected from the group consisting of: Arg, His,Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gln; Xaa15 is an amino acidselected from the group consisting of: Arg, Lys, Ala, Ser, Gly, Met, Asnand Gln; Xaa16 is an amino acid selected from the group consisting of:Ala, Gly, Ser, Asp and Asn; Xaa17 is an amino acid selected from thegroup consisting of: Ala, Asn, Ser, Ile, Gly, Val, Gln and Thr; Xaa18 isan amino acid selected from the group consisting of: His, Leu, Gln andAla; Xaa19 is an amino acid selected from the group consisting of: Pro,Gln, Leu, Asn and Ile; Xaa21 is an amino acid selected from the groupconsisting of: Trp, Phe, Tyr, His and Ile; Xaa22 is an amino acidselected from the group consisting of: Tyr and Phe; Xaa23 is an aminoacid selected from the group consisting of: Tyr and Phe; Xaa31 is anamino acid selected from the group consisting of: Glu, Asp, Gln, Asn,Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is an amino acid selected fromthe group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Glyand Val; Xaa34 is an amino acid selected from the group consisting of:Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu; Xaa35 is an amino acidselected from the group consisting of: Tyr, Trp and Phe; Xaa39 is anamino acid selected from the group consisting of: Glu, Gly, Ala, Ser andAsp; Xaa40 is an amino acid selected from the group consisting of: Glyand Ala; Xaa43 is an amino acid selected from the group consisting of:Asn and Gly; Xaa45 is an amino acid selected from the group consistingof: Phe and Tyr; and wherein said polypeptide inhibits kallikrein. 7.The method of claim 6, wherein Xaa6 is Ala.
 8. The method of claim 6,wherein Xaa7 is Phe.
 9. The method of claim 6, wherein Xaa8 is Lys. 10.The method of claim 6, wherein Xaa9 is Ala.
 11. The method of claim 6,wherein Xaa10 is Asp.
 12. The method of claim 6, wherein Xaa11 is Asp.13. The method of claim 6, wherein Xaa13 is Pro, Xaa15 is Arg, Xaa16 isAla, Xaa17 is Ala, Xaa18 is His and Xaa19 is Pro.
 14. The method ofclaim 6, wherein Xaa20 is Arg.
 15. The method of claim 6, wherein Xaa24is Asn.
 16. The method of claim 6, wherein Xaa25 is Ile.
 17. The methodof claim 6, wherein Xaa26 is Phe.
 18. The method of claim 6, whereinXaa27 is Thr.
 19. The method of claim 6, wherein Xaa28 is Arg.
 20. Themethod of claim 6, wherein Xaa29 is Gln.
 21. The method of claim 6,wherein Xaa31 is Glu.
 22. The method of claim 6, wherein Xaa32 is Glu.23. The method of claim 6, wherein Xaa34 is Ile.
 24. The method of claim6, wherein Xaa35 is Tyr.
 25. The method of claim 6, wherein Xaa39 isGlu.
 26. The method of claim 6, wherein Xaa41 is Asn.
 27. The method ofclaim 6, wherein Xaa42 is Arg.
 28. The method of claim 6, wherein Xaa44is Arg.
 29. The method of claim 6, wherein Xaa46 is Glu.
 30. The methodof claim 6, wherein Xaa47 is Ser.
 31. The method of claim 6, whereinXaa48 is Leu.
 32. The method of claim 6, wherein Xaa49 is Glu.
 33. Themethod of claim 6, wherein Xaa50 is Glu.
 34. The method of claim 6,wherein the polypeptide comprises two or more amino acids selected fromthe group consisting of: Asp at Xaa10; Asp at Xaa11; Pro at Xaa13; Argat Xaa15; Ala at Xaa16; Ala at Xaa17; His at Xaa18; Pro at Xaa19; Tip atXaa21; Phe at Xaa22; Phe at Xaa23; Glu at Xaa31; Glu at Xaa32; Ile atXaa34; Tyr at Xaa35; Glu at Xaa39; Gly at Xaa40; Asn at Xaa43; and Pheat Xaa45.
 35. The method of claim 6, wherein the polypeptide comprisesfive or more amino acids selected from the group consisting of: Asp atXaa10; Asp at Xaa11; Pro at Xaa13; Arg at Xaa15; Ala at Xaa16; Ala atXaa17; His at Xaa18; Pro at Xaa19; Trp at Xaa21; Phe at Xaa22; Phe atXaa23; Glu at Xaa31; Glu at Xaa32; Ile at Xaa34; Tyr at Xaa35; Glu atXaa39; Gly at Xaa40; Asn at Xaa43; and Phe at Xaa45.
 36. The method ofclaim 6, wherein the polypeptide comprises ten or more amino acidsselected from the group consisting of: Asp at Xaa10; Asp at Xaa11; Proat Xaa13; Arg at Xaa15; Ala at Xaa16; Ala at Xaa17; His at Xaa18; Pro atXaa19; Trp at Xaa21; Phe at Xaa22; Phe at Xaa23; Glu at Xaa31; Glu atXaa32; Ile at Xaa34; Tyr at Xaa35; Glu at Xaa39; Gly at Xaa40; Asn atXaa43; and Phe at Xaa45.
 37. The method of claim 6, wherein thepolypeptide comprises fifteen or more amino acids selected from thegroup consisting of: Asp at Xaa10; Asp at Xaa11; Pro at Xaa13; Arg atXaa15; Ala at Xaa16; Ala at Xaa17; His at Xaa18; Pro at Xaa19; Trp atXaa21; Phe at Xaa22; Phe at Xaa23; Glu at Xaa31; Glu at Xaa32; Ile atXaa34; Tyr at Xaa35; Glu at Xaa39; Gly at Xaa40; Asn at Xaa43; and Pheat Xaa45.
 38. The method of claim 6, wherein Xaa3 is Ser.
 39. The methodof claim 6, wherein Xaa2 is His.
 40. The method of claim 6, wherein Xaa1is Met.
 41. The method of claim 6, wherein Xaa56 is Thr.
 42. The methodof claim 6, wherein Xaa57 is Arg.
 43. The method of claim 6, whereinXaa58 is Asp.
 44. The method according to claim 6, wherein thekallikrein inhibitor is a polypeptide comprising amino acids 3-60 of SEQID NO:2.
 45. The method according to claim 6, wherein the kallikreininhibitor is a polypeptide comprising SEQ ID NO:2.
 46. The method ofclaim 6, wherein the organ or tissue is heart, lung, kidney, pancreas,liver, intestine, endothelial tissue, vascular tissue or skin.
 47. Amethod for reducing reperfusion injury of an organ during surgery and/orfollowing removal of the organ from a subject, comprising placing theorgan in an organ storage and preservative solution, wherein thesolution comprises a kallikrein inhibitor.
 48. The method according toclaim 47, wherein the kallikrein inhibitor is a polypeptide comprisingSEQ ID NO:1, wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 areeach individually an amino acid or absent; Xaa6, Xaa7, Xaa8, Xaa9,Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41, Xaa42, Xaa44,Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54 can be anyamino acid; Xaa10 is an amino acid selected from the group consistingof: Asp and Glu; Xaa11 is an amino acid selected from the groupconsisting of: Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr; Xaa13 is anamino acid selected from the group consisting of: Arg, His, Pro, Asn,Ser, Thr, Ala, Gly, Lys and Gln; Xaa15 is an amino acid selected fromthe group consisting of: Arg, Lys, Ala, Ser, Gly, Met, Asn and Gln;Xaa16 is an amino acid selected from the group consisting of: Ala, Gly,Ser, Asp and Asn; Xaa17 is an amino acid selected from the groupconsisting of: Ala, Asn, Ser, Ile, Gly, Val, Gln and Thr; Xaa18 is anamino acid selected from the group consisting of: His, Leu, Gln and Ala;Xaa19 is an amino acid selected from the group consisting of: Pro, Gln,Leu, Asn and Ile; Xaa21 is an amino acid selected from the groupconsisting of: Trp, Phe, Tyr, His and Ile; Xaa22 is an amino acidselected from the group consisting of: Tyr and Phe; Xaa23 is an aminoacid selected from the group consisting of: Tyr and Phe; Xaa31 is anamino acid selected from the group consisting of: Glu, Asp, Gln, Asn,Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is an amino acid selected fromthe group consisting of: Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Glyand Val; Xaa34 is an amino acid selected from the group consisting of:Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu; Xaa35 is an amino acidselected from the group consisting of: Tyr, Trp and Phe; Xaa39 is anamino acid selected from the group consisting of: Glu, Gly, Ala, Ser andAsp; Xaa40 is an amino acid selected from the group consisting of: Glyand Ala; Xaa43 is an amino acid selected from the group consisting of:Asn and Gly; Xaa45 is an amino acid selected from the group consistingof: Phe and Tyr.
 49. The method according to claim 47, wherein thekallikrein inhibitor is a polypeptide comprising amino acids 3-60 of SEQID NO:2.
 50. The method according to claim 47, wherein the kallikreininhibitor is a polypeptide comprising SEQ ID NO:2.
 51. The method ofclaim 47, wherein the organ or tissue is heart, lung, kidney, pancreas,liver, intestine, endothelial tissue, vascular tissue or skin.
 52. Acomposition for preserving and/or storing an organ comprising aphysiologically acceptable ex vivo organ preservation solutioncontaining a polypeptide comprising the amino acid sequence: Xaa1 Xaa2Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11 Gly Xaa13 Cys Xaa15 Xaa16Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23 Xaa24 Xaa25 Xaa26 Xaa27 Xaa28Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Xaa40 Xaa41Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48 Xaa49 Xaa50 Cys Xaa52 Xaa53Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO:1), wherein Xaa1, Xaa2, Xaa3,Xaa4, Xaa56, Xaa57 or Xaa58 are each individually an amino acid or isabsent; Xaa6, Xaa7, Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27,Xaa28, Xaa29, Xaa41, Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50,Xaa52, Xaa53 and Xaa54 can be any amino acid; Xaa10 is an amino acidselected from the group consisting of: Asp and Glu; Xaa11 is an aminoacid selected from the group consisting of: Asp, Gly, Ser, Val, Asn,Ile, Ala and Thr; Xaa13 is an amino acid selected from the groupconsisting of: Arg, His, Pro, Asn, Ser, Thr, Ala, Gly, Lys and Gln;Xaa15 is an amino acid selected from the group consisting of: Arg, Lys,Ala, Ser, Gly, Met, Asn and Gln; Xaa16 is an amino acid selected fromthe group consisting of: Ala, Gly, Ser, Asp and Asn; Xaa17 is an aminoacid selected from the group consisting of: Ala, Asn, Ser, Ile, Gly,Val, Gln and Thr; Xaa18 is an amino acid selected from the groupconsisting of: His, Leu, Gln and Ala; Xaa19 is an amino acid selectedfrom the group consisting of: Pro, Gln, Leu, Asn and Ile; Xaa21 is anamino acid selected from the group consisting of: Trp, Phe, Tyr, His andIle; Xaa22 is an amino acid selected from the group consisting of: Tyrand Phe; Xaa23 is an amino acid selected from the group consisting of:Tyr and Phe; Xaa31 is an amino acid selected from the group consistingof: Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile and Thr; Xaa32 is anamino acid selected from the group consisting of: Glu, Gln, Asp Asn,Pro, Thr, Leu, Ser, Ala, Gly and Val; Xaa34 is an amino acid selectedfrom the group consisting of: Thr, Ile, Ser, Val, Ala, Asn, Gly and Leu;Xaa35 is an amino acid selected from the group consisting of: Tyr, Trpand Phe; Xaa39 is an amino acid selected from the group consisting of:Glu, Gly, Ala, Ser and Asp; Xaa40 is an amino acid selected from thegroup consisting of: Gly and Ala; Xaa43 is an amino acid selected fromthe group consisting of: Asn and Gly; Xaa45 is an amino acid selectedfrom the group consisting of: Phe and Tyr; and wherein said polypeptideinhibits kallikrein.
 53. The composition of claim 52, wherein Xaa6 isAla.
 54. The composition of claim 52, wherein Xaa7 is Phe.
 55. Thecomposition of claim 52, wherein Xaa8 is Lys.
 56. The composition ofclaim 52, wherein Xaa9 is Ala.
 57. The composition of claim 52, whereinXaa10 is Asp.
 58. The composition of claim 52, wherein Xaa11 is Asp. 59.The composition of claim 52, wherein Xaa13 is Pro, Xaa15 is Arg, Xaa16is Ala, Xaa17 is Ala, Xaa18 is His and Xaa19 is Pro.
 60. The compositionof claim 52, wherein Xaa20 is Arg.
 61. The composition of claim 52,wherein Xaa24 is Asn.
 62. The composition of claim 52, wherein Xaa25 isIle.
 63. The composition of claim 52, wherein Xaa26 is Phe.
 64. Thecomposition of claim 52, wherein Xaa27 is Thr.
 65. The composition ofclaim 52, wherein Xaa28 is Arg.
 66. The composition of claim 52, whereinXaa29 is Gln.
 67. The composition of claim 52, wherein Xaa31 is Glu. 68.The composition of claim 52, wherein Xaa32 is Glu.
 69. The compositionof claim 52, wherein Xaa34 is Ile.
 70. The composition of claim 52,wherein Xaa35 is Tyr.
 71. The composition of claim 52, wherein Xaa39 isGlu.
 72. The composition of claim 52, wherein Xaa41 is Asn.
 73. Thecomposition of claim 52, wherein Xaa42 is Arg.
 74. The composition ofclaim 52, wherein Xaa44 is Arg.
 75. The composition of claim 52, whereinXaa46 is Glu.
 76. The composition of claim 52, wherein Xaa47 is Ser. 77.The composition of claim 52, wherein Xaa48 is Leu.
 78. The compositionof claim 52, wherein Xaa49 is Glu.
 79. The composition of claim 52,wherein Xaa50 is Glu.
 80. The composition of claim 52, wherein thepolypeptide comprises two or more amino acids selected from the groupconsisting of: Asp at Xaa10; Asp at Xaa11; Pro at Xaa13; Arg at Xaa15;Ala at Xaa16; Ala at Xaa17; His at Xaa18; Pro at Xaa19; Trp at Xaa21;Phe at Xaa22; Phe at Xaa23; Glu at Xaa31; Glu at Xaa32; Ile at Xaa34;Tyr at Xaa35; Glu at Xaa39; Gly at Xaa40; Asn at Xaa43; and Phe atXaa45.
 81. The composition of claim 52, wherein the polypeptidecomprises five or more amino acids selected from the group consistingof: Asp at Xaa10; Asp at Xaa11; Pro at Xaa13; Arg at Xaa15; Ala atXaa16; Ala at Xaa17; His at Xaa18; Pro at Xaa19; Trp at Xaa21; Phe atXaa22; Phe at Xaa23; Glu at Xaa31; Glu at Xaa32; Ile at Xaa34; Tyr atXaa35; Glu at Xaa39; Gly at Xaa40; Asn at Xaa43; and Phe at Xaa45. 82.The composition of claim 52, wherein the polypeptide comprises ten ormore amino acids selected from the group consisting of: Asp at Xaa10;Asp at Xaa11; Pro at Xaa13; Arg at Xaa15; Ala at Xaa16; Ala at Xaa17;His at Xaa18; Pro at Xaa19; Trp at Xaa21; Phe at Xaa22; Phe at Xaa23;Glu at Xaa31; Glu at Xaa32; Ile at Xaa34; Tyr at Xaa35; Glu at Xaa39;Gly at Xaa40; Asn at Xaa43; and Phe at Xaa45.
 83. The composition ofclaim 52, wherein the polypeptide comprises fifteen or more amino acidsselected from the group consisting of: Asp at Xaa10; Asp at Xaa11; Proat Xaa13; Arg at Xaa15; Ala at Xaa16; Ala at Xaa17; His at Xaa18; Pro atXaa19; Trp at Xaa21; Phe at Xaa22; Phe at Xaa23; Glu at Xaa31; Glu atXaa32; Ile at Xaa34; Tyr at Xaa35; Glu at Xaa39; Gly at Xaa40; Asn atXaa43; and Phe at Xaa45.
 84. The composition of claim 52, wherein Xaa3is Ser.
 85. The composition of claim 52, wherein Xaa2 is His.
 86. Thecomposition of claim 52, wherein Xaa1 is Met.
 87. The composition ofclaim 52, wherein Xaa56 is Thr.
 88. The composition of claim 52, whereinXaa57 is Arg.
 89. The composition of claim 52, wherein Xaa58 is Asp. 90.The composition according to claim 52, wherein the polypeptide comprisesSEQ ID NO:2.
 91. A method for preserving an organ or tissue comprisingcontacting the organ or tissue with a physiologically acceptable ex vivoorgan preservation solution containing a kallikrein binding polypeptide.92. The method according to claim 91, wherein the kalilkrein bindingpolypeptide comprises SEQ ID NO:2.
 93. The method of claim 91, whereinthe organ or tissue is heart, lung, kidney, pancreas, liver, intestine,endothelial tissue, vascular tissue or skin.
 94. The method of claim 91,wherein the kallikrein binding polypeptide comprises a Kunitz domain.95. The method of claim 94, wherein the Kunitz domain comprises acysteine at each of positions 5 and 55; 14 and 38; and 30 and 51, andfurther comprises: amino acid number 13 selected from His and Pro; aminoacid number 16 selected from Ala and Gly; amino acid number 17 selectedfrom Ala, Asn, and Ser; amino acid number 18 selected from His and Leu;and amino acid number 19 selected from Gln, Leu, and Pro (SEQ ID NO:23),wherein the amino acid numbering corresponds to the amino acid positionsof the Kunitz domain of bovine pancreatic trypsin inhibitor (BPTI). 96.The method of claim 94, wherein the Kunitz domain comprises a cysteineat each of positions 5 and 55; 14 and 38; and 30 and 51, and furthercomprises: amino acid number 13 selected from His and Pro; amino acidnumber 15 selected from Lys and Arg; amino acid number 16 selected fromAla and Gly; amino acid number 17 selected from Ala, Asn, and Ser; aminoacid number 18 selected from His and Leu; and amino acid number 19selected from Gln, Leu, and Pro, amino acid number 31 is Glu; amino acidnumber 32 selected from Glu and Gln; amino acid number 34 selected fromSer, Thr, and Ile; and amino acid number 39 selected from Gly, Glu, andAla (SEQ ID NO:24), wherein the amino acid numbering corresponds to theamino acid positions of the Kunitz domain of bovine pancreatic trypsininhibitor (BPTI).
 97. A method according to claim 94, wherein the Kunitzdomain is selected from the group consisting of KKII/3 #1 (SEQ ID NO:24)KKII/3 #2 (SEQ ID NO:25) KKII/3 #3 (SEQ ID NO:26) KKII/3 #4 (SEQ IDNO:27) KKII/3 #5 (SEQ ID NO:28) KKII/3 #6 (SEQ ID NO:29) KKII/3 #7 (SEQID NO:30) KKII/3 #8 (SEQ ID NO:31) KKII/3 #9 (SEQ ID NO:32) and KKII/3#10 (SEQ ID NO:33).